Andrea R. Yoder

Natriuretic Peptides: Their Structures, Receptors, Physiologic Functions and Therapeutic Applications

Natriuretic peptides are a family of three structurally related hormone/ paracrine factors. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are secreted from the cardiac atria and ventricles, respectively. ANP signals in an endocrine and paracrine manner to decrease blood pressure and cardiac hypertrophy. BNP acts locally to reduce ventricular fibrosis. C-type natriuretic peptide (CNP) primarily stimulates long bone growth but likely serves unappreciated functions as well. ANP and BNP activate the transmembrane guanylyl cyclase, natriuretic peptide receptor-A (NPR-A). CNP activates a related cyclase, natriuretic peptide receptor-B (NPR-B). Both receptors catalyze the synthesis of cGMP, which mediates most known effects of natriuretic peptides. A third natriuretic peptide receptor, natriuretic peptide receptor-C (NPR-C), clears natriuretic peptides from the circulation through receptor-mediated internalization and degradation. However, a signaling function for the receptor has been suggested as well. Targeted disruptions of the genes encoding all natriuretic peptides and their receptors have been generated in mice, which display unique physiologies. A few mutations in these proteins have been reported in humans. Synthetic analogs of ANP (anaritide and carperitide) and BNP (nesiritide) have been investigated as potential therapies for the treatment of decompensated heart failure and other diseases. Anaritide and nesiritide are approved for use in acute decompensated heart failure, but recent studies have cast doubt on their safety and effectiveness. New clinical trials are examining the effect of nesiritide and novel peptides, like CD-NP, on these critical parameters. In this review, the history, structure, function, and clinical applications of natriuretic peptides and their receptors are discussed.

A Familial Mutation Renders Atrial Natriuretic Peptide Resistant to Proteolytic Degradation

A heterozygous frameshift mutation causing a 12-amino acid extension to the C terminus of atrial natriuretic peptide (ANP) was recently genetically linked to patients with familial atrial fibrillation (Hodgson-Zingman, D. M., Karst, M. L., Zingman, L. V., Heublein, D. M., Darbar, D., Herron, K. J., Ballew, J. D., de Andrade, M., Burnett, J. C., Jr., and Olson, T. M. (2008) N. Engl. J. Med. 359, 158–165). The frameshift product (fsANP), but not wild-type ANP (wtANP), was elevated in the serum of affected patients, but the molecular basis for the elevated peptide concentrations was not determined. Here, we measured the ability of fsANP to interact with natriuretic peptide receptors and to be proteolytically degraded. fsANP and wtANP bound and activated human NPR-A and NPR-C similarly, whereas fsANP had a slightly increased efficacy for human NPR-B. Proteolytic susceptibility was addressed with novel bioassays that measure the time required for kidney membranes or purified neutral endopeptidase to abolish ANP-dependent activation of NPR-A. The half-life of fsANP was markedly greater than that of wtANP in both assays. Additional membrane proteolysis studies indicated that wtANP and fsANP are preferentially degraded by neutral endopeptidase and serine peptidases, respectively. These data indicate that the familial ANP mutation associated with atrial fibrillation has only minor effects on natriuretic peptide receptor interactions but markedly modifies peptide proteolysis.

Comparison of Polymorphisms in Genes Involved in Polycyclic Aromatic Hydrocarbon Metabolism with Urinary Phenanthrene Metabolite Ratios in Smokers

The hypothesis that interindividual differences among smokers in the metabolism of polycyclic aromatic hydrocarbons (PAH) are related to lung cancer risk has been extensively investigated in the literature. These studies have compared lung cancer risk in groups of smokers with or without polymorphisms in genes involved in PAH metabolism. We believe that carcinogen metabolite phenotyping, involving the actual measurement of PAH metabolites, would be a better way to investigate differences in lung cancer risk. With this goal in mind, we have developed methods for quantifying phenanthrene metabolites in urine. Phenanthrene is the simplest PAH with a bay region, a feature closely associated with carcinogenicity. The urinary metabolite r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) is a measure of metabolic activation, whereas phenanthrols (HOPhe) are a measure of detoxification. In this study, we quantified urinary PheT/HOPhe ratios in 346 smokers who were also genotyped for 11 polymorphisms in genes involved in PAH metabolism: CYP1A1MspI, CYP1A1I462V, CYP1B1R48G, CYP1B1A119S, CYP1B1L432V, CYP1B1N453S, EPHX1Y113H, EPHX1H139R, GSTP1I105V, GSTP1A114V, and GSTM1 null. The geometric mean molar PheT/3-HOPhe ratio was 4.08 (95% confidence interval, 3.79-4.39). Ten percent of the smokers had PheT/3-HOPhe ratios of ≥9.90. We found a significant association between the presence of the CYP1A1I462V polymorphism and high PheT/3-HOPhe ratios (P = 0.02). This effect was particularly strong in females and in combination with the GSTM1 null polymorphism. In contrast, the CYP1B1R48G and CYP1B1A119S polymorphisms were associated with significantly lower PheT/3-HOPhe ratios, particularly in Blacks. There were no consistent significant effects of any of the other polymorphisms on PheT/3-HOPhe ratios. The highest 10% of PheT/3-HOPhe ratios could not be predicted by the presence of any of the 11 polymorphisms individually or by certain combinations. The effects of the CYP1A1I462 polymorphism observed here, particularly in combination with GSTM1 null, are quite consistent with reports in the literature. However, the results of this study indicate that genotyping is not an effective way to predict PAH metabolism at least as represented by PheT/HOPhe ratios. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1805–11)

Metabolites of a tobacco-specific lung carcinogen in children exposed to secondhand or thirdhand tobacco smoke in their homes

Background: People exposed to secondhand tobacco smoke (SHS) inhale the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which is metabolized to NNAL and its glucuroniders. These urinary metabolites, termed total NNAL, can be quantified. A related compound, iso-NNAL, has been proposed as a biomarker for exposure to smoke constituent residues on surfaces (thirdhand tobacco smoke). There is limited information in the literature on levels of total NNAL in children exposed to SHS. Methods: We recruited 79 parent–child dyads from homes where the enrolled parent was a cigarette smoker and visited their homes. Parents were asked questions, home ambient air quality was evaluated, and children provided urine samples. Urine was analyzed for total NNAL, total cotinine, total nicotine, and iso-NNAL. Results: Ninety percent of the children had detectable total NNAL in urine; total nicotine and total cotinine were also detected in most samples. There were significant positive relationships between biomarker levels and exposure of children in the home. Levels were highest in homes with no smoking restrictions. African American children had significantly higher levels than other children. iso-NNAL was not detected in any urine sample. Conclusions: There was nearly universal exposure of children to the lung carcinogen NNK, due mainly to exposure to SHS from adult smokers in their homes. Impact: Homes with adult smokers should adopt restrictions to protect their children from exposure to a potent lung carcinogen. Cancer Epidemiol Biomarkers Prev; 20(6); 1213–21. ©2011 AACR.

Longitudinal study of urinary phenanthrene metabolite ratios: effect of smoking on the diol epoxide pathway.

We have proposed that urinary phenanthrene metabolites could be used in a carcinogen metabolite phenotyping approach to identify individuals who may be susceptible to cancer induction by polycyclic aromatic hydrocarbons (PAH). In support of this proposal, we have developed methods for quantitation of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and phenanthrols (HOPhe) in human urine. PheT is the end product of the diol epoxide metabolic activation pathway of PAH, whereas HOPhe are considered as detoxification products. In this study, we investigated the longitudinal consistency of these metabolites over time in smokers and nonsmokers and compared their levels. Twelve smokers and 10 nonsmokers provided urine samples daily for 7 days, then weekly for 6 weeks. Levels of PheT, HOPhe, and PheT/HOPhe ratios were relatively constant in most individuals, with mean coefficients of variation ranging from 29.3% to 45.7%. There were no significant changes over time in levels of the metabolites or in ratios. These results indicate that a single urine sample should be sufficient when comparing phenanthrene metabolites in different groups. PheT/HOPhe ratios were significantly higher in smokers than in nonsmokers, showing that smoking induces the diol epoxide metabolic activation pathway of phenanthrene. This finding is consistent with previous studies indicating that inducibility of PAH metabolism contributes to cancer risk in smokers. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2969–74)

Temporal stability of urinary and plasma biomarkers of tobacco smoke exposure among cigarette smokers.

Intraindividual variability of measurements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), nicotine, cotinine, and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) over time is uncertain. From 70 habitual smokers’ plasma and urine sampled bimonthly for a year we analysed plasma for NNAL, cotinine and PheT, and urine for NNAL, cotinine and nicotine. We estimated the intraclass correlation coefficients (ρI) for each measurement. Plasma and creatinine-corrected urinary NNAL were stable (ρI ≥70%); plasma PheT and plasma and urinary total cotinine were fairly stable (ρI ≥50%), but urinary nicotine ρI ≈ 40% was not. Except for nicotine, single measurements from plasma or urine adequately represent individual mean exposure over time.

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab-/- mice

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologues. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487), and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529), and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild-type, S487A, and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.

A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.

Determination of new phosphorylation sites within natriuretic peptide receptors using mass spectrometric methods

Background While it has long been know that phosphorylation of the kinase homology domain is a key regulatory mechanism of natriuretic peptide receptors, no biochemical proof for individual phosphorylation sites has been reported. Here, we describe biochemical verification of previously identified phosphorylation sites in both natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) as well as the identification of novel phosphorylation sites in both receptors.